Promising antimicrobials from Phoma spp.: progress and prospects

The increasing multidrug-resistance in pathogenic microbes and the emergence of new microbial pathogens like coronaviruses have necessitated the discovery of new antimicrobials to treat these pathogens. The use of antibiotics began after the discovery of penicillin by Alexander Fleming from Penicillium chrysogenum. This has attracted the scientific community to delve deep into the antimicrobial capabilities of various fungi in general and Phoma spp. in particular. Phoma spp. such as Phoma arachidicola, P. sorghina, P. exigua var. exigua, P. herbarum, P. multirostrata, P. betae, P. fimeti, P. tropica, among others are known to produce different bioactive metabolites including polyketides, macrosporin, terpenes and terpenoids, thiodiketopiperazines, cytochalasin derivatives, phenolic compounds, and alkaloids. These bioactive metabolites have already demonstrated their antimicrobial potential (antibacterial, antifungal, and antiviral) against various pathogens. In the present review, we have discussed the antimicrobial potential of secondary metabolites produced by different Phoma species. We have also deliberated the biogenic synthesis of eco-friendly antimicrobial silver nanoparticles from Phoma and their role as potential antimicrobial agents.


Introduction
There are terrifying global reports of the multidrugresistance in pathogens that are not responding to the available antibiotics (Wencewicz 2019). The main reasons for developing resistance by microbes include misuse and overuse of antibiotics, and environmental factors (Ghosh et al. 2020;Christaki et al. 2020). This problem of antibiotic resistance has garnered the attention of the scientific community, policymakers, and the public at large from all over the world, and it is a global health challenge (Markley and Wencewicz 2018;Hu et al. 2021).
The new and emerging diseases caused by microbes are major threat to mankind. The recent emergence of the COVID-19 pandemic caused by SARS-CoV-2 is a burning example that has devastated human life globally. The current burden of co-infections and superinfections such as mucormycosis in COVID-19 patients is also a great issue that emphasizes the discovery of new antimicrobials (Feldman and Anderson 2021). Moreover, there has been huge concern about re-emerging microbial diseases such as malaria, tuberculosis, influenza, cholera, pertussis, etc.

Open Access
*Correspondence: mahendrarai@sgbau.ac.in Unfortunately, for more than three decades, no new antibiotics have been discovered (Böttcher et al. 2021), and therefore, these facts warrant the discovery of new antibiotics and/ or search for new alternatives from natural products such as plants and microbes to tackle such a grave problem (WHO Newsletter 2020). Among the microbes, fungi play a key role in the production of antimicrobials. The serendipitous discovery of penicillin by Alexander Fleming (1929) from Penicillium notatum and P. chrysogenum is the best example (Zhu et al. 2014). Other potential antibiotics produced by fungi include cephalosporins and griseofulvin. Several species of Phoma such as P. arachidicola, P. sorghina, P. exigua var. exigua, P. herbarum, P. multirostrata, P. betae, and P. fimeti are pigment-producing (Chande et al. 1899) and some Phoma species have already demonstrated the antimicrobial potential against various fungi (Aoyagi et al. 2007;Hussain et al. 2014), bacteria (Huang et al. 2017;Chen et al. 2019) and viruses (Liu et al. 2019;Peng et al. 2020). They produce secondary metabolites with antimicrobial potential. These bioactive compounds include polyketides like anthraquinones and diphenyl ether derivatives; ergocytochalasin A, macrosporin, thiodiketopiperazines, cytochalasin derivatives, and alkaloids. The antimicrobial metabolites producing species of Phoma can be harnessed to treat various microbial pathogens.
The present review is focused on the antimicrobial potential of secondary metabolites produced by different terrestrial, marine or endophytic Phoma species. Moreover, the biogenic synthesis of eco-friendly antimicrobial silver nanoparticles produced from Phoma and their role as potential antimicrobial agents have been discussed. The review is timely as so far there is no review available on the antimicrobial nature of metabolites produced by different Phoma species.

Phoma: the producer of novel bioactive metabolites
The Phoma spp. are widely distributed as pathogens of plants, animals, and humans, and also in soil, water and air (Rai 2002). The Phoma spp. secrete various metabolites that have already demonstrated antimicrobial potential (Rai et al. 2009a(Rai et al. , b, c, 2018Herath et al. 2009). Not only terrestrial but marine and endophytic species of Phoma are also responsible for the production of antimicrobial metabolites (Hoffman et al. 2008;Bhimba et al. 2012;Elsebai et al. 2016Elsebai et al. , 2018. A large number of metabolites with unique structures, and potential biological and pharmacological activities have been reported from the marine Phoma species particularly P. sorghina, P. herbarum, and P. tropica. These metabolites generally include lactones, quinine, diterpenes, phthalate, enolides, and anthraquinones ( Fig. 1a-c). which have shown a broad range of bioactivities including antimicrobial, anticancer, radical scavenging, and cytotoxic (Rai et al. 2018(Rai et al. , 2020. There are several reports which provide conclusive evidence that endophytic Phoma species living in plants secrete potential antimicrobial compounds (Fig. 2) (Hussain et al. 2015;Huang et al. 2017;da Silva et al. 2017;de Vries et al. 2018;Nalli et al. 2019;El-Zawawy et al. 2020;Li et al. 2020;Rai et al. 2020;Hu et al. 2021). For example, the compounds like α-tetralone derivative (3S)-3,6,7trihydroxy-α-tetralone, together with cercosporamide, β-sitosterol, and trichodermin reported from the ethyl acetate extract of endophytic Phoma sp. (ZJWCF006) isolated from Arisaema erubescens (Wang et al., 2012). These compounds were found to be effective against the plant pathogenic fungi such as Fusarium oxysporum, Rhizoctonia solani, Colletotrichum gloeosporioides, Magnaporthe oryzae, and plant pathogenic bacteria including Xanthomonas campestris and X. oryzae.
Many species of Phoma have demonstrated remarkable antimicrobial activities. For example, Hussain et al. (2014) isolated phomafuranol (I), phomalacton (II), (3R)-5-hydroxymellein (III), and emodin (IV) (Fig. 3) from the ethyl acetate fractions of Phoma spp. recovered from Fucus serratus. which demonstrated potential inhibitory activities including antibacterial, antifungal, and antialgal. Arora et al. (2016) screened endophytes isolated from Glycyrrhiza glabra and reported the presence of Phoma spp. which was closely related to P. cucurbitacearum. Further, the authors isolated two thiodiketopiperazine derivatives (Fig. 4) from the extract of this species of Phoma which showed remarkable antibacterial activity against Staphylococcus aureus and S. pyogene Moreover, these compounds significantly inhibited the biofilm formation ability of both the pathogens singly and in combination with ciprofloxacin and ampicillin in a synergistic way. Endophytic Phoma spp. (URM 7221) isolated from the leaves of Schinus terebinthifolius effectively inhibited S. aureus, MRSA, B. subtilis, and E. faecalis (de Silva  In another study, Chen et al. (2019) reported that Phoma species SYSU-SK-7 inhabiting endophytically in mangrove plant Kandelia candel contains polyketides that have shown significant activity against Pseudomonas aeruginosa, Staphylococcus aureus followed by Candida albicans. Recently, Peng and his colleagues (2020) reported ergocytochalasin A from P. multirostrata which was found as an endophyte in Parasenecio albus. The bioactive compound demonstrated strong activity against different pathogenic viruses including Human dengue virus type 3 (DV3), influenza A virus (H1N1), and respiratory syncytial virus (RSV).
Several members of the genus Phoma are well-known to produce a wide range of antimicrobials that are specific to the target organisms (bacteria, fungi, and viruses). P. exigua var. exigua produces antibiotic E and cytochalasin B (Boerema and Howeler 1967), P. pigmentivora produces LL-D253alpha (McIntyre et al. 1984), P. lingam (Tode) Desm. yields phomenoic acid and phomenolactone which are antibacterial and antifungal compounds (Topgi et al. 1987). In addition, there are other bioactive compounds reported from Phoma spp. For example, a well-known anti-infective agent squalestatin was reported from a Phoma spp. (Dawson et al. 1992); antitumor compound fusidienol A from another Phoma spp. (Singh et al. 1997), andYamaguchi et al. (2002) isolated the bioactive compound FOM-8108 which inhibited neutral sphingomyelinases.

Biosynthesis of silver nanoparticles by Phoma spp. and its antimicrobial efficacy
As discussed earlier, Phoma species are known to produce a wide range of metabolites that have already shown antimicrobial activity (Rai et al. 2009a). Some  of the metabolites may not directly reveal the antimicrobial potential but can be used for the fabrication of silver nanoparticles (AgNPs) which also demonstrated remarkable antimicrobial potential. AgNPs are well known as a new generation of antimicrobials (Rai et al., 2009b). An elaborative account of multiple modes of action of AgNPs is reviewed by Dakal et al. (2016) and a schematic representation of the same is given in Fig. 6.  Phoma sp. Phoma species are capable of extracellular synthesis of spherical AgNPs and silver nanorods. Extracellular synthesis of nanoparticles by Phoma spp. offer an advantage of obtaining large quantities of AgNPs at a rapid rate and in a relatively pure state. Furthermore, the extracellular synthesis of AgNPs by Phoma spp. would make the process simple and easier for downstream processing; fungal broths can be easily filtered by filter press of similar simple equipment, thus making it a cost-effective process (Gade et al., 2010). Moreover, the fabrication of AgNPs by Phoma spp. is a green and eco-friendly approach as no toxic chemicals, high temperature, or pressure are used for the synthesis (Gade et al., 2014;Rai et al. 2021a, b).
In a study, the fabrication of AgNPs by P. glomerata (MTCC-2210) was reported by Birla et al. (2009). Authors also reported the combined activity of commercial antibiotics and AgNPs synthesised from Phoma spp. by testing against E. coli JM-103 (ATCC-39403) and S. aureus (ATCC-25923) on Muller-Hinton agar plates. Commercial antibiotics like ampicillin (10 µg), gentamycin (10 µg), kanamycin (30 µg), streptomycin (10 µg) and vancomycin (30 µg) were used in the study. The comprehensive fold increases in area were observed for ampicillin, streptomycin, and vancomycin. Thus, the combined activity observed was better in E. coli than S. aureus. Whereas the disc diffusion analysis of only AgNPs showed better activity against S. aureus as compared to E. coli. In another study, the AgNPs synthesised from P. gardinae (ITCC 4554) showed antimicrobial activity against human pathogenic bacteria and fungi (Rai et al., 2015a). Authors evaluated the activity of AgNPs against C. albicans, S. choleraesuis, P. aeruginosa, S. aureus, and E. coli. The AgNPs were found to be most effective against E. coli followed by S. aureus, C. albicans, S. choleraesuis, and P. aeruginosa as compared with antibiotics. Further extracellular synthesis of AgNPs by P. capsulatum, P. putaminum, and P. citri was reported by Rai and co-workers (2015b). The AgNPs syjthesised from these Phoma spp. showed potential antimicrobial activity against Aspergillus niger, C. albicans, S. choleraesuis, P. aeruginosa, S. aureus, and E. coli. The least minimal inhibitory concentration (MIC) of 0.85 μg/ml was shown by AgNPs synthesized from P. citri against S. choleraesuis. AgNPs fabricated using Phoma spp. is not only reported for antibacterial and antifungal activity but also demonstrated antiviral potential. Some Phoma spp. isolated from the infected plants and identified on the basis of morphological and molecular characteristics were used for the fabrication of AgNPs. Fig. 6 The four most prominent routes of antimicrobial action of AgNPs. 1. AgNPs adhere to microbial cell surface and results in membrane damage and altered transport activity; 2. AgNPs penetrate inside the microbial cells and interact with cellular organelles and biomolecules, and thereby, affect respective cellular machinery; 3. AgNPs cause increase in ROS inside the microbial cells leading to cell damage and; 4. AgNPs modulate cellular signal system ultimately causing cell death. Reproduced from Dakal et al. (2016)

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This demonstrated a significant decrease in replication efficiency for Herpes Simplex Virus (HSV)-1 and human parainfluenza virus (HPIV) type-3, and a minor effect on the replication of HSV-2 at a concentration of 10 mg/ml (Gaikwad et al. 2013). Further, the authors reported that AgNPs ability to control viral infectivity was most likely attributed to the size and zeta potential of the fabricated AgNPs, which interfere with virus and cell interaction, thereby blocking viral entry into the cell. Shende et al. (2017) synthesised AgNPs using immobilized biomass of the P. exigua var. exigua. This process was found to be a simple, fast, large-scale, and efficient route for the synthesis of AgNPs, without disintegration of calcium alginate beads in the medium for ten batch cycles. The immobilization of P. exigua biomass leads to the development of a method for the continuous synthesis of AgNPs. Moreover, this large-scale synthesis process could be a boon to the commercial fabrication of AgNPs which will be required due to the application of AgNPs in a large number of commercial products. The AgNPs thus produced also demonstrated antibacterial activity against E. coli and S. aureus. Graphical illustration of P. exigua var. exigua biomass immobilization process and AgNPs fabrication is given in Fig. 7.
It is evident from the above reports that the different Phoma spp. possess tremendous ability to reduce the inorganic metal ions to nanoparticles in general and AgNPs in particular, which is due to the extracellular secretions of metabolites by Phoma spp. Consequently, the metabolites secreted by the Phoma spp. can be harnessed and explored for the synthesis of nanoparticles of different sizes and shapes. In near future, the possibility of utilizing antimicrobial metabolites secreted by Phoma spp. for the fabrication of AgNPs cannot be overlooked, since these metabolites can be used with AgNPs synergistically which will provide the solution to the increasing drug resistance problem worldwide.

Conclusions
Antimicrobial resistance and the entry of new fatal microbes like Coronavirus have made the researchers to seriously think about searching for new strategies to combat the global problem. Thus, there is a high demand for new antibiotics for difficult-to-treat bacteria and other Fig. 7 Graphical illustration of Phoma exigua var. exigua biomass immobilization process and AgNPs fabrication (Shende et al. 2017), reprinted with permission pathogenic microbes. In this context, various fungi including Phoma offers antimicrobial metabolites. Various species of Phoma particularly pigment-producing species such as P. arachidicola, P. sorghina, P. exigua var. exigua, P. herbarum, P. multirostrata, P. betae, and P. fimeti have already demonstrated their potential against pathogenic fungi, bacteria, and viruses. Moreover, several species of Phoma have been studied for the production of bioactive compounds such as polyketides, ergocytochalasin A, macrosporin, thiodiketopiperazines, terpenes, terpenoids, and alkaloids which have shown their antimicrobial potential. These antimicrobial metabolites of Phoma spp. are not only terrestrial but also include marine and endophytic spp. dwelling in medicinal plants. Moreover, some Phoma species are also known to synthesize silver nanoparticles extracellularly which have already proven to be the new generation of antimicrobials. Such a process of nanoparticle synthesis is eco-friendly, economically viable and a greener approach without the use of harmful chemicals and high pressure and temperature. These nanoparticles can also be utilized as nanocarriers for the slow and sustained delivery of antimicrobial drugs. Finally, more thorough research is required to screen different species of Phoma from extreme environments to find out potential antibiotic producers.